Attempts to induce broadly reactive and potent protective anti-HIV antibodies (Abs) by immunization of humans have been unsuccessful. The optimal vaccine-induced Abs are usually considered to be neutralizing Abs that have been defined by studies of several human monoclonal Abs (mAbs) as targeting epitopes in the membrane proximal external region of gp41 and in various regions of gp120 including the CD4 binding site, a complex of high mannose residues, the bridging sheet, and the V3 loop. However, HIV-infected individuals are able to produce both neutralizing Abs that target additional epitopes and non-neutralizing Abs that block virus replication through pathways other than that mediated by pH-independent virus/cell fusion. These latter non-neutralizing Abs inhibit viral replication through a variety of mechanisms such as those mediated by Fc receptor-bearing cells and by complement. Based on these findings, we propose to focus efforts on identifying new protective human mAbs, i.e., both conventional neutralizing mAbs specific for previously unrecognized envelope (Env) epitopes, and human mAbs that inhibit virus replication via non-neutralizing pathways. For this, hybridomas will be generated from cells of US and Cameroon subjects infected with HIV strains carrying clade B and A Envs, respectively. Three new screening methods will be used for mAb selection, and a variety of methods will be employed to test the ability of the new mAbs to inhibit virus replication. The work is divided into three aims: 1) Identification of new neutralizing mAbs which target previously unrecognized epitopes, and selection of mAbs with virus inhibitory activities that function through non-neutralizing pathways. Pseudoviruses, Env-expressing cells and gp120 reagents will be used in several screening assays, each chosen to maximize the different types and cross-reactivity of mAbs to be selected, and to determine if there are epitopes that are uniquely or preferentially targeted by newly transmitted viruses. 2) Delineation of the inhibitory activities of these mAbs, of their breadth against viruses and pseudoviruses from multiple clades, and of the different patterns of reactivity between the clade B- and A- derived mAbs. These comparisons, together with descriptions of the immunologic nature and specificities of the mAbs, will provide the first comprehensive and systematic information about the characteristics of mAbs with different protective activities. And, 3) Mapping of the epitopes targeted by the new mAbs. Immunologic, virologic and physicochemical techniques will be used to delineate mAb epitopes, the specific interactions between mAb and Env, and quaternary Env structures targeted by mAbs. The reagents generated and the knowledge gained will illuminate mechanisms of virus neutralization and pathways that interrupt virus replication, and will inform the design of reagents for use in active immunization (vaccines), passive immunization, and immunotherapy.